Tissue fractionation studies. 13. Analysis of mitochondrial fractions from rat liver by density-gradient centrifuging

Abstract

Mitochondrial fractions from rat liver have been analyzed by means of density-gradient centrifuging. Two types of experiments were performed. In one, sedimentation boundaries and the derived sedimentation-coefficient distribution curves were determined after incomplete sedimentation in a stabilizing gradient. In the other, solutions of high density were used to make the gradient and the particles were allowed to approach density equilibrium with the medium by prolonged centrifuging at high speed. In each case, the distributions of up to 9 different enzymes were studied simultaneously. Cytochrome oxidase, glutamic dehydrogenase, malic dehydrogenase and alkaline deoxyribonuclease showed practically identical distributions under all conditions applied. The bulk of the proteins of the preparation showed the same distribution pattern, which undoubtedly was that of the true mitochondria. These particles appeared to be remarkably homogeneous with respect to their enzymic content. The 5 lysosomal hydrolases, acid phosphatase, B-glucuronidase, cathepsin, acid ribonuclease and acid deoxyribonuclease, formed another distinct group, characterized by a lower range of sedimentation coeffcients, a greater dispersion of density and a higher average density, especially in heavy water, than the mitochondria. The distribution curves of these enzymes were similar about not identical, indicating that if lysosomes form a single species, they are not enzymically homogeneous. Uricase showed many properties in common with the lysosomal enzymes, but differed from them by having sharper distribution curves and a particularly large increase in equilibrium density in heavy water.